Báo cáo khoa học: An E3 ubiquitin ligase, Synoviolin, is involved in the degradation of immature nicastrin, and regulates the production of amyloid b-protein doc

An E3 ubiquitin ligase, Synoviolin, is involved in the
degradation of immature nicastrin, and regulates the
production of amyloid b-protein
Tomoji Maeda
1
, Toshihiro Marutani
2,
*, Kun Zou
1
, Wataru Araki
3
, Chiaki Tanabe
1
, Naoko Yagishita
4
,
Yoshihisa Yamano
4
, Tetsuya Amano
4
, Makoto Michikawa
2
, Toshihiro Nakajima
4,5,6
and
Hiroto Komano
1
1 Department of Neuroscience, School of Pharmacy, Iwate Medical University, Morioka, Japan
2 Department of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology, Aichi, Japan
3 Department of Demyelinating Disease and Aging, National Institute of Neuroscience, Tokyo, Japan
4 Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan

5 Choju Medical Institute and Fukushimura Hospital, Toyohashi, Japan
6 Misato Marine Hospital, Kochi, Japan
Introduction
Amyloid b-protein (Ab), which is the major compo-
nent of senile plaques in the brains of patients with
Alzheimer’s disease, is generated from the amyloid pre-
cursor protein (APP) through its sequential proteolytic
Keywords
amyloid b-protein; E3 ubiquitin ligase;
nicastrin; presenilin; c-secretase
Correspondence
H. Komano, Department of Neuroscience,
School of Pharmacy, Iwate Medical
University, 2-1-1 Nishitokuta, Yahaba,
Shiwa, Iwate 028-3694, Japan
Fax: +81 19 698 1864
Tel: +81 19 651 5111, extn 5210
E-mail:
*Present address
Department of Biology, Faculty of Sciences,
Kyushu University Graduate School,
Fukuoka, Japan
(Received 13 January 2009, revised 2
August 2009, accepted 6 August 2009)
doi:10.1111/j.1742-4658.2009.07264.x
The presenilin complex, consisting of presenilin, nicastrin, anterior pharynx
defective-1 and presenilin enhancer-2, constitutes c-secretase, which is
required for the generation of amyloid b-protein. In this article, we show
that Synoviolin (also called Hrd1), which is an E3 ubiquitin ligase implicated
in endoplasmic reticulum-associated degradation, is involved in the degrada-

tion of endogenous immature nicastrin, and affects amyloid b-protein genera-
tion. It was found that the level of immature nicastrin was dramatically
increased in synoviolin-null cells as a result of the inhibition of degradation,
but the accumulation of endogenous presenilin, anterior pharynx defective-1
and presenilin enhancer-2 was not changed. This was abolished by the
transfection of exogenous Synoviolin. Moreover, nicastrin was co-immuno-
precipitated with Synoviolin, strongly suggesting that nicastrin is the substrate
of Synoviolin. Interestingly, amyloid b-protein generation was increased by
the overexpression of Synoviolin, although the nicastrin level was decreased.
Thus, Synoviolin-mediated ubiquitination is involved in the degradation of
immature nicastrin, and probably regulates amyloid b-protein generation.
Structured digital abstract
l
MINT-7255352: Synoviolin (uniprotkb:Q9DBY1) physically interacts (MI:0915) with NCT
(uniprotkb:
P57716)byanti tag coimmunoprecipitation (MI:0007)
l
MINT-7255377: Ubiquitin (uniprotkb:P62991) physically interacts (MI:0915) with NCT (uni-
protkb:
P57716)byanti bait coimmunoprecipitation (MI:0006)
l
MINT-7255363: NCT (uniprotkb:P57716) physically interacts (MI:0915) with Synoviolin (uni-
protkb:
Q9DBY1)byanti bait coimmunoprecipitation (MI:0006)
Abbreviations
Ab, amyloid b-protein; APH-1, anterior pharynx defective-1; APP, b-amyloid precursor protein; CTF, C-terminal fragment; ER, endoplasmic
reticulum; NCT, nicastrin; NTF, N-terminal fragment; PEN-2, presenilin enhancer-2; PS, presenilin.
5832 FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS
cleavage catalyzed by b- and c-secretases [1]. b-Secre-
tase has been identified as a membrane-tethered aspar-

tyl protease [2]. c-Secretase activity is attributed to the
presenilin (PS) complex, which is composed of four
transmembrane proteins: PS, nicastrin (NCT), preseni-
lin enhancer-2 (PEN-2) and anterior pharynx defec-
tive-1 (APH-1) (collectively named PS cofactors in this
study) (reviewed in [3]). Full-length PS is endoproteo-
lytically processed into two fragments: the N-terminal
fragment (NTF) and the C-terminal fragment (CTF)
[4]. The processed PS resides in the c-secretase complex
(reviewed in [3]). Endogenous PS, NCT, PEN-2 and
APH-1 are mainly localized in the endoplasmic reticu-
lum (ER) and Golgi [5], and the properly assembled
complex is transported through the secretory pathway
to localize predominantly in the Golgi and then at the
cell surface [6,7].
NCT is a type I transmembrane protein that
possesses many potential glycosylation sites within its
large ectodomain [8]. Several studies have established
that three principal forms of NCT exist in cells: the
unglycosylated, nascent protein ( 80 kDa); an imma-
ture N-linked glycosylated species (immature NCT,
 110 kDa); and a mature N-linked isoform (mature
NCT,  150 kDa) which is formed after entering the
Golgi apparatus [9]. The mature NCT associates with
active c-secretase [10] and, importantly, PS is
required for the full post-translational generation of
this mature NCT species [9]. In addition, NCT is cri-
tical for the stability and trafficking of other c-secre-
tase components, and NCT affects Ab production
[11].

Interestingly, the cellular level of PS is tightly limited
[12]. Excess PS cofactors which fail to reside in the
complex, such as full-length PS, mostly undergo ubi-
quitin ⁄ proteasome-mediated degradation, although the
precise mechanism of elimination of excess cofactors is
not fully understood [12].
Ubiquitination is required for proteasome-mediated
degradation, although, recently, accumulating evidence
has shown that ubiquitin has multiple functions,
including intracellular trafficking (reviewed in [13]),
which is accomplished through the sequential actions
of enzymes: an activating enzyme (E1), a conjugating
enzyme (E2) and a ligase (E3) (reviewed in [14]). Of
the three enzymes, E3 enzymes are the key determining
factors in substrate protein selection. Synoviolin, a
representative of ER-resident E3 ubiquitin ligase, is a
mammalian homolog of yeast Hrd1 [15]. Synoviolin is
also a pathogenic factor in rheumatoid arthritis [16],
and is involved in ER-associated degradation [17]. The
substrates of Synoviolin were found to include polyglu-
tamine-expanded huntingtin [18], the tumor suppressor
gene p53 [19] and Parkin-associated endothelin recep-
tor-like receptor [20].
In this study, we addressed whether Synoviolin is
involved in the degradation of PS cofactors using syno-
violin-null cells, as PS cofactors undergo the ubiqui-
tin ⁄ proteasome pathway. We report that Synoviolin is
involved in the degradation of immature NCT and reg-
ulates Ab generation.
Results

Accumulation of immature NCT in synoviolin-null
cells
To investigate whether Synoviolin is involved in the
degradation of PS cofactors, we first compared the
levels of PS cofactors by immunoblotting between
synoviolin-null cells and wild-type (wt) cells. As shown
in Fig. 1, the level of endogenous immature NCT was
found to be markedly increased in synoviolin-null cells,
compared with wt cells, although endogenous PS,
APH-1 and PEN-2 were not changed in synoviolin-null
cells. Interestingly, the mobilities of immature and
mature NCT on the gel in synoviolin-null cells were
slightly faster than that in wt cells (Fig. 1A). This is
probably a result of the difference in the degree of
sugar modification, because deglycosylation treatment
of NCT in synoviolin-null cells resulted in a similar
mobility to that in wt cells (Fig. S1, see Supporting
Information). We also determined the levels of c-secre-
tase-unrelated ER protein (calnexin) and cytoskeleton
protein (tubulin) in these cells as the internal control
proteins. The calnexin and tubulin levels were found to
be similar between these cells, confirming that the same
amount of protein was loaded in each lane (Fig. 1A).
In addition, the observed accumulation of endogenous
immature NCT in synoviolin-null cells was abolished
by exogenously expressed Synoviolin, but not by the
expression of Synoviolin C307A mutant lacking E3
ubiquitin ligase activity [21], indicating that the lack of
E3 ubiquitin ligase activity of Synoviolin causes the
accumulation of immature NCT (Fig. 1B, right panel).

As shown in Fig. 1B (left panel), the overexpression of
Synoviolin in wt cells decreased both immature and
mature NCT levels; however, very interestingly, the
expression of Synoviolin C307A mutant in wt cells
caused the accumulation of much more immature
NCT than mature NCT. Because the C307A mutant
inhibits the ubiquitination mediated by endogenous
Synoviolin in a dominant-negative manner, as reported
previously [21], this result strongly suggests that Syno-
violin-mediated ubiquitination is involved in the
preferential degradation of immature NCT.
T. Maeda et al. Synoviolin is involved in the degradation of nicastrin
FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS 5833
Effect of Synoviolin on the stability of NCT
Because Synoviolin is an E3 ubiquitin ligase for pro-
teasome-dependent protein degradation, it is most
likely that the accumulation of NCT in synoviolin-null
cells is a result of the suppression of the degradation
of NCT. To further investigate this, we next com-
pared the degradation of NCT with time between
synoviolin-null cells and wt cells. As shown in Fig. 2,
western blot analysis of the intracellular degradation
of NCT in synoviolin-null cells and wt cells following
cycloheximide treatment revealed that immature NCT
in synoviolin-null cells remained stable, as did mature
NCT, although, in wt cells, the immature NCT level
was preferentially decreased at 10 h after treatment.
As a decrease in the immature NCT level seems to
include effects of both its maturation and degrada-
tion, we further confirmed the degradation of imma-

ture NCT in wt cells with treatment by the
proteasome inhibitor MG-132. As shown in Fig. 2C,
the treatment of wt cells with MG-132 was found to
preferentially increase the level of immature NCT
compared with that of mature NCT, strongly suggest-
ing that immature NCT is preferentially degraded by
the proteasome. Taken together, Synoviolin is most
likely to be involved in the preferential degradation
of immature NCT via the ubiquitin ⁄ proteasome
pathway.
Synoviolin interacts with NCT
E3 ligases for ubiquitination confer specificity to the
ubiquitin system by directly interacting with the sub-
strate proteins and helping to transfer ubiquitin to
them. Therefore, to determine whether NCT is the
substrate of Synoviolin, we determined whether Syno-
violin interacts with NCT. As shown in Fig. 3, imma-
ture NCT was coimmunoprecipitated with anti-FLAG
IgG and, in addition, Synovolin was coimmunopreci-
pitated with anti-NCT IgG when FLAG-tagged Syno-
violin and NCT were coexpressed in synoviolin-null
cells. These results indicate that Synoviolin interacts
19
25
37
PS 1
CT F
115
im NC T
(kDa )

115
82
Ca l
Tu b
48
64
30
20
Syno
–/– +/+
Syno
–/– +/+
Syno
–/– +/+
Syno
Syno
–/– +/+
+/+
AP H 1aL
20
14
PEN-2
mN CT
Transgen e :
115
(kDa )
mNCT
imNC T
α -tub
64

Syno (–/–)
(–/–)
Sy no ––
Syno (+/+ )
C 307A
Sy no
C 307A
A
B
Fig. 1. Accumulation of immature NCT in synoviolin-null fibroblasts. (A) The components of the PS complex (NCT, PS-1, APH-1, PEN-2) in
the lysate (20 lg) of synoviolin-null fibroblasts were detected by immunoblotting with anti-NCT IgG, anti-APH1aL IgG and anti-PEN-2 IgG.
Calnexin and a-tubulin in the lysate were also immunodetected as internal markers. ) ⁄ ), synoviolin-null fibroblasts; + ⁄ +, wt fibroblasts. (B)
NCT in the lysates from wt fibroblasts (left panel) and synoviolin-null fibroblasts (right panel), retrovirally expressing Synoviolin or Synoviolin
C307A mutant lacking E3 ubiquitin ligase activity, was detected by immunoblotting with anti-NCT IgG. Mutation of the conserved cysteine
307 to alanine in Synoviolin disrupts its ligase activity and this C307A mutant functions in a dominant-negative manner [21]. a-Tubulin in the
lysate was also detected as internal marker. imNCT, immature NCT; mNCT, mature NCT; –, mock transfection; Syno, Synoviolin; C307A,
Synoviolin C307A mutant; a-tub, a-tubulin.
Synoviolin is involved in the degradation of nicastrin T. Maeda et al.
5834 FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS
with immature NCT. In addition, the degree of ubiqui-
tination of NCT in wt cells was also found to be
slightly higher than that in synoviolin-null cells
(Fig. 3C). However, it was also noted that NCT was
slightly ubiquitinated even in synoviolin-null cells.
Therefore, it is most likely that NCT is a substrate of
Synoviolin, but the other E3 ubiquitin ligase also
appears to ubiquitinate NCT.
Cycloheximide (h)
0610
Syno (+/+)

Syno (–/–)
0610
0
50
100
0246810
115
(kDa)
mNCT
imNCT
64
α-tub
Remaining %
Time (h)
*
*
*
mNCT, Syno (–/–)
imNCT, Syno (–/–)
mNCT, Syno (+/+)
imNCT, Syno (+/+)
115
(kDa)
mNCT
imNCT
MG-132
-
A
BC
Fig. 2. Degradation of NCT in synoviolin-null and wt fibroblasts. (A) synoviolin-null and wt fibroblasts were treated with 20 lgÆmL

)1
cyclohex-
imide and harvested at the times indicated. NCT in RIPA-solubilized lysates (10 lg) was detected by immunoblotting with anti-NCT antibody.
a-Tubulin in the lysates was also immunodetected as an internal control for a stable protein. Each sample was duplicated. imNCT, immature
NCT; mNCT, mature NCT; a-Tub, a-tubulin. (B) The intensities of the bands corresponding to immature NCT and mature NCT in (A) were
densitometrically quantified using a luminescent image analyzer LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). NCT levels remaining at
each time point were calculated as a percentage of the intensity at time zero. Each value is the average of four independent experiments.
Asterisk indicates significant differences from time zero [significant difference at P < 0.05 (Student’s t-test)]. (C) Wt fibroblasts were treated
with 10 l
M MG-132 for 10 h, and NCT in the RIPA-solubilized lysates (10 lg) was detected by immunoblotting with anti-NCT antibody.
–, cells treated without MG-132.
im
NC T
180
115
(kDa )
Cell
lysate
Ig G A nti-flag
IP :
75
Cell
lysate
Ig G A nti-NC T
IP :
Syno
115
180
IP :
Ig G U b I gG Ub

WB :
N

C
T
Ubiquitinated NCT
Syno (–/–) Syno (+/+ )
m
NC T
A B
C
Fig. 3. Synoviolin interacts with NCT. (A)
The cell lysates of synoviolin-null fibroblasts
transiently coexpressing FLAG-tagged Syno-
violin and NCT were immunoprecipitated
with anti-FLAG antibody and immunode-
tected with anti-NCT antibody. (B) The same
cell lysates were immunoprecipitated with
anti-NCT antibody and then immunode-
tected with anti-Synoviolin antibody. (C)
After synoviolin-null and wt fibroblasts tran-
siently transfected with NCT had been trea-
ted with cycloheximide and lactacystin for
8 h, the cells were harvested. The RIPA-
solubilized lysates (1 mg) were immunopre-
cipitated with anti-ubiquitin mouse antibody
(mouse IgG for control) and then immunode-
tected with anti-NCT antibody. IP, immuno-
precipitation; WB, western blot.
T. Maeda et al. Synoviolin is involved in the degradation of nicastrin

FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS 5835
Detection of NCT on the cell surface in
synoviolin-null cells
Only mature NCT goes to the cell surface, and immature
NCT stays within the cells, as reported previously [6,22].
As the level of immature NCT was greatly increased and
the molecular weight of NCT was changed slightly in
synoviolin-null cells, we investigated whether the cellular
localization of NCT was different between synoviolin-
null cells and wt cells. To determine this, we detected
NCT localized at the plasma membrane in synoviolin-
null cells. For this purpose, we labeled the cell surface
proteins with biotin, and then detected the surface-bioti-
nylated NCT by immunoblotting with anti-NCT IgG.
As shown in Fig. 4A, we found that both immature and
mature NCT were clearly detected on the cell surface in
synoviolin-null cells, although, in wt cells, only mature
NCT was detected on the cell surface. In addition, the
mature NCT level on the cell surface was increased in
synoviolin-null cells (Fig. 4B) [percentage of mature
NCT at the cell surface relative to that in the total lysate:
24% (wt) versus 64% (Syn) ⁄ ))]. These results indicate
that a functional deletion of Synoviolin causes a change
in the intracellular trafficking of NCT.
Effect of Synoviolin on the production of Ab
NCT is one of the essential cofactors of the c-secretase
complex. We therefore investigated the effect of the
Synoviolin-mediated degradation of NCT on Ab gen-
eration. In Fig. 5, we measured the Ab level secreted
from wt fibroblasts overexpressing APP [23]. As shown

in Fig. 5A, B, the overexpression of Synoviolin
enhanced the production of Ab40 and Ab42 by about
twofold, whereas the secretion of soluble APP was not
changed in these cells. Figure 5C also showed that the
endogenous NCT level was decreased and the intracel-
lular APP level was not changed by the overexpression
of Synoviolin. Previously, the targeting of NCT to the
cell surface enhanced Ab generation, because one of
the main Ab generation sites is likely to be in the cell
surface [6]. Therefore, it is possible that the overex-
pression of Synoviolin enhances the localization of
NCT at the cell surface, resulting in an enhancement
of Ab generation. To test this possibility, we measured
the level of NCT on the cell membrane. No increase
in the cell surface NCT level in cells overexpressing
Synoviolin was observed (Fig. 5D).
Discussion
In this study, we showed that Synoviolin is involved in
the intracellular degradation of NCT. Of the four
c-secretase components, only NCT was found to be
degraded by Synoviolin. In addition, Synoviolin
appears to preferentially target immature NCT for
degradation, because synoviolin-null cells exhibited the
accumulation of immature NCT, and the expression of
the dominant-negative Synoviolin mutant lacking E3
ubiquitin ligase activity in wt cells caused a greater
accumulation of immature NCT than mature NCT.
115
82
(kDa

A
B
)
Ce ll
ly s ate
Ce
ll
me
mb ra
ne
Ce ll
ly
s
ate
Ce ll
me
mb
ra ne
Syno (+/+ ) Syno (–/–)
imNC T
mNCT
115
mIntegrin β1
imIntegrin β1
37
elF3 f
0
10
20
30

40
50
60
70
mNCT in the cell membrane
(% of the cell lysate)
Syno (+/+) Syno (–/–)
Fig. 4. Cell surface distribution of immature and mature NCT in
synoviolin-null fibroblasts. (A) Cell surface proteins of synoviolin-null
and wt fibroblasts were biotinylated as described in Materials and
methods. The lysates of surface-biotinylated cells were then incu-
bated with streptavidin–agarose. Total lysate (20 lg) and biotiny-
lated proteins (streptavidin–agarose bound) were immunodetected
with anti-NCT IgG, anti-integrin b1 IgG (as a control for the cell
surface protein) [22] and anti-elF3f IgG (as a control for the cyto-
solic protein) [32]. imNCT, immature NCT; mNCT, mature NCT; m
integrin b1, mature integrin b1; im integrin b1, immature integrin
b1. (B) Band intensities were densitometrically quantified with a
luminescent image analyzer LAS-3000 (Fuji Photo Film Co., Ltd.),
and the percentage mature NCT level in the cell membrane relative
to that in the total cell lysate was calculated. Data are the average
of two independent experiments. The percentage immature NCT
level in the cell membrane relative to that in the total cell lysate in
synoviolin-null cells was 22.0 ± 4.5%.
Synoviolin is involved in the degradation of nicastrin T. Maeda et al.
5836 FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS
Interestingly, the sugar modification of NCT in
synoviolin-null cells appeared to be slightly different
from that in wt cells. This may suggest that Synovi-
lin-mediated ubiquitination also regulates the traffick-

ing of NCT within the Golgi compartment, because
the maturation of the sugar modification of the pro-
tein occurs within the Golgi compartment. Recently,
there has been an expansion of the recognized roles
for ubiquitin in processes other than proteasome-
dependent proteolysis, which includes intracellular
trafficking (reviewed in [13]). In this regard, it is note-
worthy that both immature NCT and mature NCT
delivered to the cell surface were increased in synovio-
lin-null cells, although only the mature form of NCT
goes to the cell surface in wt cells (Fig. 4). It appears
that Synoviolin somehow suppresses the direct deliv-
ery of NCT from ER to the cell surface. Previously,
it has been shown that Synoviolin increases the mem-
brane localization of huntingtin protein [18], also sug-
gesting that Synoviolin is involved in intracellular
trafficking.
We also found that NCT interacts with Synoviolin
(Fig. 3), strongly suggesting that NCT is the substrate
of Synoviolin. As reported previously, NCT undergoes
ubiquitination [24]. We found that the degree of ubi-
quitination of NCT in wt cells was higher than that in
synoviolin-null cells. Therefore, NCT is most likely to
be a substrate of Synoviolin. However, the other E3
ubiquitin ligase also appears to ubiquitinate NCT,
because NCT was ubiqutinated slightly even in syno-
violin-null cells. Indeed, in synoviolin-null cells, NCT
started to degrade more than 10 h after cycloheximide
treatment (data not shown). Further study of the
mechanism underlying NCT degradation mediated by

Synoviolin, including an in vitro study, is needed.
It was also noted that the overexpression of Syno-
violin increased the Ab level, whereas the cellular level
of NCT decreased in transfected cells, because a
decreased NCT level would be expected to decrease
the Ab level. Because the levels of full-length APP and
soluble APP were not changed (Fig. 5), it is likely that
c-cleavage was increased. As reported previously [25],
0
1
2
3
0
200
400
600
800
1000
Aβ (pmol·mg
–1
total protein)
Sy ––
–
no Sy no
Aβ40
Aβ42
sAPP (ng·mg
–1
total protein)
Syno

*
*
P
Transgene:
Transgene:
Aβ level
sAPP level
0
20
40
60
80
Syno–
Ce
ll
lysate
Cell
membrane
Ce
ll

ly
sate
Cell
membrane
Syno–
115
(kDa)
Transgene:
Cell surface NCT

imNCT
mNCT
115
mIntegrin β1
imIntegrin β1
37
elF3f
mNCT in the cellm embrane
(% of the cell lysate)
A

–
180
115
82
(kDa)
AP
180
115
82
(kDa)
Transgene:
Syno
–
Syno
Transgene:
NCT and intracellularAPP
imNCT
mNCT
C

B

D
Fig. 5. Effect of the overexpression of Synoviolin on Ab generation. Wild-type murine fibroblasts expressing APP were retrovirally expressed
with Synoviolin. Ab (A) and soluble APP (B) secreted from the cells during a 96-h culture were detected by ELISA. Values are the mean-
s ± SEM of four independent dishes (n = 4). Asterisk indicates significant differences from mock [significant difference at P < 0.01
(Student’s t-test)]. (C) NCT and intracellular APP in the cell lysates were immunodetected with anti-NCT IgG and 22C11 respectively. (D) Left
panel: cell surface NCT in the mock- or Synovilin-transfected cells was immunodetected as described in Fig. 3. Integrin b1 (as a control for
the cell surface protein) and eIF3f (as a control for the cytosolic protein) were also immunodetected. Similar results were obtained from
three independent experiments. m integrin b1, mature integrin b1; im integrin b1, immature integrin b1. Right panel: the band intensities were
quantified, and the percentage mature NCT level in the cell membrane relative to that of the total cell lysate is shown. Data are the average
of three independent experiments. –, mock transfection; Syno, Synoviolin transfection; imNCT, immature NCT; mNCT, mature NCT.
T. Maeda et al. Synoviolin is involved in the degradation of nicastrin
FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS 5837
the cell membrane NCT level is thought to be more
important than the intracellular level of NCT for Ab
generation. Therefore, we investigated whether Syno-
violin enhances the cell surface localization of NCT;
however, no increase in the cell membrane NCT level
in cells transfected with Synoviolin was observed. It
has also been shown that the overexpression of SEL-
10, that is an E3 ligase for PS1 ubiquitination, causes
a decrease in the level of PS1, but an increase in Ab
secretion [26]. This suggests that SEL-10-mediated ubi-
quitination modulates the PS1 complex in APP proces-
sing, although the exact mechanism is not known.
Therefore, likewise, Synoviolin-mediated ubiquitination
can also regulate Ab generation, possibly through the
modulation of intracellular trafficking. As the over-
expression of Synoviolin was suggested to increase

c-cleavage, as mentioned above, the overexpression of
Synoviolin, probably through ubiquitination, could
promote the trafficking of the PS complex to the site
at which c-cleavage occurs, or activate c-secretase
itself.
In this study, we conclude that Synoviolin is
involved in the degradation of immature NCT. We
have also shown that the expression of Synoviolin
enhances Ab generation. Further study of the mechan-
ism underlying the enhancement of Ab generation by
Synoviolin will clarify the interaction between the ubi-
qutination of the PS complex and APP processing.
Materials and methods
Antibodies, reagents and cell lines
A mouse anti-PS1 monoclonal IgG (for the CTF of PS1)
was purchased from Chemicon International (Temecula,
CA, USA). A rabbit anti-NCT IgG and a mouse NCT
monoclonal IgG were purchased from Sigma (St. Louis,
MO, USA) and Chemicon International, respectively. MG-
132 was purchased from Sigma. A rabbit anti-APH1aL
antibody was purchased from COVANCE (Berkeley, CA,
USA). Anti-PEN-2 IgM was provided by Dr Thinakaran
[27,28]. Anti-a-tubulin and anti-calnexin IgG were pur-
chased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA, USA). Anti-APP N-terminal antibody 22C11 was pur-
chased from Sigma. Anti-HRD1 (Synoviolin) C-terminal
antibody was purchased from ABGENT (San Diego CA,
USA). Anti-elF3f was purchased from Rockland Inc.
(Gilbertsville, PA, USA). Anti-integrin b1 antibody was
purchased from BD Biosciences (San Jose, CA, USA).

Monoclonal antibody against mono- and polyubiquitin
was purchased from BIOMOL (Plymouth Meeting, PA,
USA). Synoviolin-null murine fibroblasts [29] and murine
fibroblasts overexpressing human APP were cultured in
Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure
Chemical Industries, Ltd., Osaka, Japan) containing 10%
fetal bovine serum.
Plasmids and retrovirus-mediated infection
PMX-Synoviolin was generated as described previously
[16]. cDNA encoding Synoviolin C307A mutant was gener-
ated by overlap PCR using the following primers:
5¢-AAATGTGGTTGGCGGGCAGTCTCTTGGC-3¢ and
5¢-ACTGCCCGCCAACCACATTTTCC-3¢. The PCR pro-
duct was verified by sequencing. The retrovirus-mediated
infection was carried out as reported previously [30].
Cycloheximide treatment
Cells (5 · 10
5
) plated on 60 mm tissue culture dishes were
grown for 24 h; cycloheximide was then added to a final
concentration of 20 lgÆmL
)1
. At various times after the
addition of cycloheximide, the cells were harvested and
lysed in RIPA buffer (150 mm NaCl, 10 mm Tris ⁄ HCl pH
7.5, 1% Nonidet P-40, 0.1% SDS and 0.2% sodium deoxy-
cholate) containing a protease inhibitor cocktail.
Immunoprecipitation, immunoblotting and ELISA
Cultured cells were lysed in RIPA buffer containing a pro-
tease inhibitor cocktail. The solubilized proteins were sub-

jected to immunoprecipitation as described previously [31].
The precipitated proteins were resolved by SDS-PAGE on
4–20% gel for the detection of PS and NCT. Immunoblot-
ting was performed as reported previously [31]. ELISAs for
Ab and soluble APP were performed using a bAmyloid
ELISA kit (Wako Pure Chemical Industries, Ltd., Osaka,
Japan) and human soluble APP ELISA kit (IBL Co., Ltd.,
Nagoya, Japan), respectively.
Cell surface biotinylation
Cell surface biotinylation was carried out using a cell sur-
face protein isolation kit (Pierce, Rockford, IL, USA). The
cells were grown in four 10 cm tissue culture dishes, and
washed twice with ice-cold NaCl ⁄ P
i
. The cells were incu-
bated in 10 mL of ice-cold sulfosuccinimidy-2-(biotina-
mido)-ethyl-1,3-dithiopropionate (0.25 mgÆmL
)1
) in ice-cold
NaCl ⁄ P
i
for 30 min at 4 °C, and then 500 lL of the
quenching solution were added to each dish to quench the
reaction. The cells were scraped and washed twice with
Tris-buffered saline (TBS) (10 mm Tris ⁄ HCl pH 7.5,
150 mm NaCl) and lysed in lysis buffer containing protease
inhibitors. Each lysate was incubated with streptavidin–
agarose beads at 4 °C for 60 min, and the captured proteins
were eluted with 50 mm dithiothreitol in Laemmli’s SDS
sample buffer.

Synoviolin is involved in the degradation of nicastrin T. Maeda et al.
5838 FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS
Acknowledgements
We thank Dr Gopal Thinakaran for providing anti-
PEN-2 IgG. This study was supported in part by a
grant-in-aid for scientific research from the Ministry of
Education, Culture, Sports, Science and Technology of
Japan, and by a grant from the Ministry of Health,
Labor and Welfare of Japan. We thank Dr Paul Lang-
man for assistance with the English.
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Supporting information
The following supplementary material is available:
Fig. S1. Deglycosylation of NCT.
This supplementary material can be found in the
online version of this article.
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Synoviolin is involved in the degradation of nicastrin T. Maeda et al.
5840 FEBS Journal 276 (2009) 5832–5840 ª 2009 The Authors Journal compilation ª 2009 FEBS